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Distribution of 14C-labelled ochratoxin a in pregnant mice
Fd Chem. Toxic. Vol. 21, No. 5, pp. 563-568, 1983 Printed in Great Britain. All rights reserved
0278-6915/83$3.00+ 0.00 Copyright ~C 1983PergamonPress Ltd
D I S T R I B U T I O N OF 14C-LABELLED O C H R A T O X I N A IN P R E G N A N T MICE L.-E. APPELGREN Department of Pharmacology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, BMC, Box 573, S-751 23 Uppsala
and R. G. ARORA Department of Pathology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Box ?028, S-750 07 Uppsala, Sweden (Received 16 November 1982)
Abstract--Autoradiography was used to study the distribution of t4C-labelledochratoxin A for up to 4 hr after its iv administration to mice at various stages of pregnancy. The highest ~4C concentration was consistently found in the bile throughout the experimental period. The concentration of radioactivity in the tissues was found, in decreasing order, in the liver, kidney, blood, salivary glands, large vessels, brown fat, myocardium, uterus and lymphatic tissues. The toxin was shown to cross the placental barrier on day 9 of pregnancy, at which time it is most effective in producing foetal malformations.
INTRODUCTION
EXPERIMENTAL
Ochratoxin A (OCT A) is a toxic metabolite produced by several species of storage moulds of the genera Aspergillus and Penicillium (for review, see Chu, 1974). The toxin has been shown to be the major cause of porcine and avian nephropathy (Elling, Hald, Jacobsen & Krogh, 1975; Krogh, Hald & Pedersen, 1973) and is suspected as a possible factor in Balkan (endemic) nephropathy in humans. The literature summarized by Hood (1979) as well as our own studies (Arora, 1982; Arora & Fr616n, 1981; Arora, Fr61en & Nilsson, 1981) have shown that the compound, besides causing tissue damage in mature animals, is potentially teratogenic, especially for mice. However, it is not teratogenic in pigs and has not been shown to cross the placental barrier in this species (Shreeve, Patterson, Pepin et al. 1977). Similarly, Munro, Scott, Moodie & Willes (1973) found no effect in ovine foetuses even with a dose that was sufficient to cause death of the dam; the toxin was found in foetal blood only in trace amounts. Information on the distribution and excretion pattern of OCT A is limited. Studies in rats by previous workers have demonstrated accumulation of the toxin in the kidney and gastro-intestinal tissues (Chang & Chu, 1977; Lillehoj, Kwolek, Elling & Krogh, 1979; Suzuki, Satoh & Yamazaki, 1977). The autoradiographic study reported here was an attempt to gain information on the general distribution of radioactivity from ~4C-labelled OCT A in the maternal and foetal tissues of mice. A preliminary report of the results obtained was presented at the International Conference on Veterinary Pharmacology, Toxicology and Therapeutics held at Cambridge in July/August, 1980.
Abbreviations: OCT A = Ochratoxin A; OCT ct =ochratoxin c~; TLC = thin-layer chromatography.
Autoradiography. Seven C57B1 female mice (obtained from the Swedish National Defence Research Institute, Stockholm) were used for whole-body autoradiography two on day 9, four on day 10 and one on day 17 of pregnancy. The day following the night of conception, as judged from vaginal plugs, was called day 1 of gestation. The animals were killed 20 rain, 1, 3 or 4 hr after injection of labelled OCT A. Longer time intervals were not possible because of the toxicity of the dose given (see below). A further four females, one each on pregnancy days 8, 9, l0 and 17, were used for autoradiography of their dissected uteri with a survival time of 20 min after injection. [~4C]OCT A prepared by fermentation using sodium [1-~4C]acetate, was generously supplied by Dr E. B. Lillehoj, SEA, USDA, New Orleans, USA. Each of the animals was injected iv with 4.8 mg [~4C]OCT A, which corresponded to about 120~170mg/kg. The substance was dissolved in 0.2 ml 0.1 M-NaHCO3. The dose used was several times the oral LDs0, which has been found to be 29 30 mg/kg (Arora, 1982). The specific activity of the labelled OCT A was 1 x 106 dpm/mg, so that every animal received about 2#Ci (E. B. Lillehoj, personal communication). Whole-body autoradiography and autoradiography of the uteri were performed according to Ullberg (1954 & 1977) after rapid freezing of the whole animals or the dissected uteri. Fluorography. Some of the sections from the isolated uteri were taken on tape 688 (3M Co., St Paul, MN, USA) and placed under ultraviolet (UV) light. The intense light-blue fluorescence of OCT A was easily distinguishable from the natural dim-blue fluorescence of the control sections from animals free from OCT A. Liquid scintillation counting and chromatograph),. One C57B1 mouse was killed on each of days 8, 9, 10 and 17 of pregnancy, 20min after injection of
563
L.-E. APPELGRENand R. G. ARORA
564
4.8 mg [14C]OCT A. The tissue samples (liver, kidney, uterus, placenta and foetus) were weighed, homogenized and extracted with 0.01 M-H3PO 4 and with chloroform. Aliquots from the water and chloroform phases were counted in a liquid scintillation counter using Aquasol 2 (NEN, Boston, MA, USA) as scintillation medium. Thin-layer chromatography (TLC) was used to analyse the chromatographic behaviour of the radioactivity in the chloroform extracts. Benzene acetic acid (9:1, v/v) was used as the solvent system and the radioactivity was detected with the aid of a radiochromatogram scanner. The non-radioactive standard OCT A was identified by its fluorescence under UV light. For a control experiment another t4C-labelled OCT A preparation was synthesized. The side-chain of non-radioactive OCT A was split off by acid hydrolysis and ~4C-labelled phenylalanine (sp. act. 30 mCi/mmol) was introduced into the molecule. The synthesis was kindly performed by Dr Bj6rn Lindgren of the Swedish National Defence Research Institute, UmeA. Since the exchange of radioactive OCT A was very small, only one animal could be used. Thus, a 9-day pregnant CBA mouse was injected iv with all the [~4C]OCT A, corresponding to 0.05/tCi. (The specific activity is not known since it was impossible to weigh the small amount. Radiochromatography of the original compound showed only one radioactive spot with the same R~ value as the reference non-radioactive OCT A.) After 20 min, the animal was killed and tissue samples were taken. The samples were counted in a liquid scintillation counter after dissolving the samples in Soluene-350 (Packard), and addition of Insta-Fluor (Packard). Quench-correction was made using an external standard. RESULTS
A utoradiography With a 20-min delay after injection of [14C]OCT A, the highest concentration of radioactivity was found in the bile, indicating rapid biliary excretion of the mycotoxin and/or its labelled metabolites (Fig. 1). The liver showed a fairly high t4C concentration as did the pelvic part of the kidney. The salivary glands and large blood vessels showed intermediate concentrations, and the brown fat, myocardium and uterus showed slightly higher concentrations than the blood. One hour after injection, the bile still had the highest ~4C concentration, followed by the liver and kidney; a "spotty" pattern was observed in the kidney cortex (Fig. 2). The blood showed a fairly high ~4C concentration which exceeded that of the myocardium and salivary glands. In the spleen and thymus the ~4C concentration was lower than in the blood. Four hours after injection, the intestinal contents (bile) showed a very high ~4C concentration, while the liver, kidney, salivary glands and brown fat showed about the same concentration as that of the blood which was still relatively high. At this time after the injection the ~4C concentration in the thymus, the white pulp of the spleen and the corpora lutea in the ovary also reached that of the blood (Fig. 3). In the bone and central nervous system no radioactivity could be detected.
Uterus and foetus With a 20-min delay after injection of labelled OCT A to the 8- and 9-day pregnant mice, the ~4C concentration in the uterine wall was higher than that of the blood (Fig. 4a,c). A still higher concentration was seen in the ectoplacental cone and in some embryonal structures most probably corresponding to the allantois and the gut, in the 9-day pregnant mice. The corresponding 10-day pregnant animals showed less concentration in the uterine wall (Fig. 4b, d) and the placental structures, but in some embryos a high concentration of radioactivity could be detected in the allantois. In the 17-day pregnant animals, the concentration was very low in the foetuses, indicating that very small amounts of radioactivity passed the placental barrier (Fig. 5a, b). High amounts of radioactivity were found, however, in the chorio-allantoic placenta and still higher amounts in the visceral yolk sac.
Fluorograpby The blue fluorescence distribution of OCT A showed the same pattern in the pregnant uteri and embryo/foetus as was shown by autoradiography (Fig. 4). The concentration of mycotoxin in the 9-day pregnant uterus was much higher than that in the 10-day pregnant uterus; some embryonal structures showed fluorescence in the 9- but not in the 10-day pregnant mice. In the 17-day pregnant uterus the strongest fluorescence was found in the visceral yolk sac placenta.
Liquid scintillation counting and chromatography The results of the radioactivity counts in the chloroform extracts of the tissues are presented in Fig. 6, showing counts in the liver, kidney and uterus 20 min after injection of [~4C]OCT A in 8-, 9- and 10-day pregnant animals. From the 17-day pregnant animal, the placenta and foetus were analysed instead of the uterus. (The ~4C concentration in the water extracts was not above the background level in the tissue samples analysed.) There was good agreement regarding the relative concentration between these results and the autoradiographic findings. The radioactivity concentration in the uteri of the 8- and 9-day pregnant animals was higher than in the 10-day pregnant uterus. In the foetus of the 17-day pregnant animal only a small amount of ~4C was found (Fig. 6). Radiochromatography of the chloroform extracts revealed that the radioactivity in the blood as well as in the liver, kidneys, foetuses and uteri (Fig. 7) corresponded to the reference substance [~4C]OCT A. In the control experiment, using the [~4C]OCT A with theoretically much higher specific activity, the highest concentration was found in the bile, followed by the blood (Table 1). DISCUSSION
Because of the low specific activity of the compound used and the minimum dose required for autoradiographic experiments, the toxicity of OCT A did not allow the animals to survive longer than 4 hr after injection. The highest concentration of radio-
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Fig. 2. Detail of whole-body [‘4C]OCT A, showing
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autoradiogram the “spotty”
Heart
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Intestinal contents
(bile)
of a 17.day pregnant mouse 1hr after iv injection distribution of radioactivity in the kidney cortex.
Fig. 3. Whole-body autoradiogram of a IO-day pregnant showing a blood concentration of ‘%I comparable
Uterus
blood
pulp of spleen
mouse 4 hr after iv injection of [‘%I]OCT A, to that of liver, kidney and spleen.
Uterus
Embryo
of
Embryo
Fig. 4. OCT A fluorescence (intense light areas) in uteri taken from mice injected 20 min earlier with c. 150 mg [‘4C]OCT A/kg on (a) day 9 and (b) day IO of pregnancy. The blue fluorescence is apparent in some embryos and is much stronger in the uterus in (a) than in (b), in which it is restricted to the uteri and placentae. This distribution pattern is reflected in the autoradiograms of adjacent sections from the (c) 9-day and (d) IO-day uteri. 565
Liver : :
Fig. 5. Detail of (a) whole-body
Foetus
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: :
autoradiogram and (b) corresponding I hr after iv injection of [‘%]OCT 566
section of a 17.day pregnant A.
mouse
Distribution of ochratoxin A
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Fig. 6. Radioactivity in chloroform extracts from the liver, kidney and uterus (placentae and foetuses) 20 min after iv Origin : ~, ! injection of ['4C]OCT A to mice at different stages of pregnancy, showing a differencein concentration in the uteri Fig. 7. Radioactive thin-layer chromatograms scanned in a between day 8 or 9 and day l0 of pregnancy (cf. Fig. 4). radiochromatogram scanner and showing one main radioactivity spot corresponding to the reference [~4C]OCTA in the chloroform extracts from livers, kidneys, uteri, placentae and tbetuses of mice injected with ['4C]OCT A. activity was found in the bile, indicating a rapid and high biliary excretion. In earlier studies, high concentrations of OCT A have been found mainly in the intestines and kidney and no biliary excretion of the toxin has been mentioned (Galtier, 1974; Lillehoj et al. 1979; Suzuki et al. 1977). Apparently, the high biliary excretion observed in the autoradiograms was not due to the very high amount of toxin administered since in our control experiment, using a tracer dose, a similar excretion pattern was observed (Table 1). Also, the concentration of ]4C in the other tissues investigated in this control experiment was of the same relative magnitude as in the autoradiograms. The blood showed a fairly high concentration 4hr after injection, a finding in agreement with the affinity of OCT A for serum albumin (Chang & Chu, 1977; Chu, 1971). The amounts of radioactivity found in the white pulp of the spleen and in the thymus 4hr after iv injection of [~4C]OCT A may be related to the lymphocidal action of OCT A in these organs (Arora, 1982; Kanisawa, Suzuki, Kozuka & Yamazaki, 1977). The "spotty" appearance of OCT A in the kidney can be related to the lesions observed in the proximal tubules (Elling et al. 1975) and to the demonstration of OCT A by immunofluorescence in the kidneys of pigs and rats (Elling, 1977). The relatively low uptake of OCT A by the uterus of the 10-day pregnant mouse compared to the 9-day animal, observed from the autoradiography as well as the fluorography studies (Fig. 4) was also confirmed by the liquid scintillation data, which showed the 10-day uterus to have considerably lower counts than either the 8- or 9-day uterus. The demonstration of a relatively high concentration of radioactivity and of a strong blue fluorescence in embryos following injection on day 9 but not in those exposed on day 10 or 17 of gestation is in agreement with the observed high frequency of malformations in foetuses of dams given OCT A on day 9 of gestation (Arora & Fr616n, 1981). The low concentration of radioactivity registered in the embryo/foetus after injection
on day 10 or 17 supports the theory that the amount crossing the placental barrier on or after day 10 of development is perhaps insufficient to induce morphological abnormalities (Arora & Fr616n, 1981). Earlier studies showed that the toxin did not pass the placental barrier in swine (Shreeve et al. 1977) and it was found only in trace amounts in the foetal blood after iv injection into pregnant sheep (Munro et al. 1973). In the present experiment the slight radioactivity found in the corpora lutea 4hr after injection can be correlated with interference of OCT A with steroidogenesis, as reported from histochemical studies in rats (Gupta, Bandyopadhyay, Mazumdar & Paul, 1980). Even though the radioactivity demonstrated in the autoradiograms could be due to metabolites of the injected compound, the chromatographic studies have shown that the radioactivity in the investigated organs corresponded to the reference substance [~4C]OCT A. Similar results were obtained in rats dosed ip, since OCT A but not its metabolite was found in the blood, liver and kidney (Nel & Purchase, 1968). However, studies using [14C]OCT A have shown that urine and faeces from orally dosed rats contained OCT A and ochratoxin ~ (OCT ~) (Suzuki et al. 1977). Homogenates of pancreas, duodenum and ileum, but not liver and kidney, have been shown Table 1. t¢C concentration in various organs and body fluids from a 9-day pregnant mouse Tissue/fluid Bile Blood Uterus Kidney Liver Muscle (skeletal)
14C concentration (dpm/g tissue × 10 3) 34.0 5.5 1.7 2.5 2.6 0.3
The animal was killed 20 min after iv injection of 0.05 #Ci [14C]OCT A (theoretical sp. act. 30 mCi/mmol).
L.-E. APPELGRENand R. G. ARORA
568
by the same authors to hydrolyse OCT A to OCT ~, indicating that this t r a n s f o r m a t i o n is probably greater in orally dosed animals than in parenterally injected animals. Acknowledgements--This work was supported by the Swedish Council for Forestry and Agricultural Research (SJFR) and the Swedish Council for Planning and Coordination of Research (heavy equipment). REFERENCES
Arora R. G. (1982). Some observations on the pathological effects of ochratoxin A in adult and foetal mice (Abstract). Toxicon 20, Suppl. 1, p. 3. Arora R. G. & Fr61~n H. (1981). Interference of mycotoxins with prenatal development of the mouse. II. Ochratoxin A induced teratogenic effects in relation to the dose and stage of gestation. Acta vet. scand. 22, 535. Arora R. G., Fr616n H. & Nilsson A. (1981). Interference of mycotoxins with prenatal development of the mouse. I. Influence of aflatoxin B~, ochratoxin A and zearalenone. Acta vet. stand. 22, 524. Chang F. C. & Chu F. S. (1977). The fate of ochratoxin A in rats. Fd Cosmet. Toxieol. 15, 199. Chu F. S. (1971). Interaction of ochratoxin A with bovine serum albumin. Archs Biochem. Biophys. 147, 359. Chu F. S. (1974). Studies on ochratoxins. C R C Crit. Rev. Toxicol. 2, 499. Elling F. (1977). Demonstration of ochratoxin A in kidneys of pigs and rats by immunofluorescence microscopy. Acta path. microbiol, scand. Sect A 85, 151. Elling F., Hald B., Jacobsen Chr. & Krogh P. (1975). Spontaneous toxic nephropathy in poultry associated with ochratoxin A. Acta path. microbiol, scand. Sect. A 83, 739. Galtier P. (1974). Devenir de l'ochratoxine A dans
l'organisme animal. II. Distribution tissulaire et 61imination chez le rat. Annls Rech V~t. 5, 319. Gupta M., Bandyopadhyay S., Mazumdar S. K. & Paul B. (1980). Ovarian steroidogenesis in rats following ochratoxin A treatment. Toxic. appl. Pharmac. 53, 515. Hood R. D. (1979). Effects of mycotoxins on development. In Advances in the Study o f Birth Defects. Vol. II. Edited by T. V. N. Persaud. p. 191. MTP Press, Lancaster. Kanisawa M., Suzuki S., Kozuka Y. & Yamazaki M. (1977). Histopathological studies on the toxicity of ochratoxin A in rats. I. Acute oral toxicity. Toxic. appl. Pharmac. 42, 55. Krogh P., Hald B. & Pedersen E. J. (1973). Occurrence of ochratoxin A and citrinin in cereals associated with mycotoxic porcine nephropathy. Aeta path. microbiol. scand. Sect. B 81, 689. Lillehoj E. B., Kwolek W. F., Elling F. & Krogh P. (1979). Tissue distribution of radioactivity from ochratoxin A~4C in rats. Mycopathologia 68, 175. Munro I. C., Scott P. M., Moodie C. A. & Willes R. F. (1973). Ochratoxin A--Occurrence and toxicity. J. Am. vet. Med. Ass. 163, 1269. Nel W. & Purchase I. F. H. (1968). The fate of ochratoxin A in rats. J. S. Afr. Chem. Inst. 21, 87. Shreeve B. J., Patterson D. S. P., Pepin G. A., Roberts B. A. & Wrathall A. E. (1977). Effects of feeding ochratoxin to pigs during early pregnancy. Br. vet. J. 133, 412. Suzuki S., Satch T. & Yamazaki M. (1977). The pharmacokinetics of ochratoxin A in rats. Jap. J. Pharmac. 27, 735. Ullberg S. (1954). Studies on the distribution and fate of 35S-labelled benzyl-penicillin in the body. Acta radioL Suppl. 118, p. 1. Ullberg S. (1977). The technique of whole body autoradiography. Cryosectioning of large specimens. The L K B Instrument Journal. Special issue on whole body autoradiography, Sweden. p. 1.
0278-6915/83$3.00+ 0.00 Copyright ~C 1983PergamonPress Ltd
D I S T R I B U T I O N OF 14C-LABELLED O C H R A T O X I N A IN P R E G N A N T MICE L.-E. APPELGREN Department of Pharmacology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, BMC, Box 573, S-751 23 Uppsala
and R. G. ARORA Department of Pathology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Box ?028, S-750 07 Uppsala, Sweden (Received 16 November 1982)
Abstract--Autoradiography was used to study the distribution of t4C-labelledochratoxin A for up to 4 hr after its iv administration to mice at various stages of pregnancy. The highest ~4C concentration was consistently found in the bile throughout the experimental period. The concentration of radioactivity in the tissues was found, in decreasing order, in the liver, kidney, blood, salivary glands, large vessels, brown fat, myocardium, uterus and lymphatic tissues. The toxin was shown to cross the placental barrier on day 9 of pregnancy, at which time it is most effective in producing foetal malformations.
INTRODUCTION
EXPERIMENTAL
Ochratoxin A (OCT A) is a toxic metabolite produced by several species of storage moulds of the genera Aspergillus and Penicillium (for review, see Chu, 1974). The toxin has been shown to be the major cause of porcine and avian nephropathy (Elling, Hald, Jacobsen & Krogh, 1975; Krogh, Hald & Pedersen, 1973) and is suspected as a possible factor in Balkan (endemic) nephropathy in humans. The literature summarized by Hood (1979) as well as our own studies (Arora, 1982; Arora & Fr616n, 1981; Arora, Fr61en & Nilsson, 1981) have shown that the compound, besides causing tissue damage in mature animals, is potentially teratogenic, especially for mice. However, it is not teratogenic in pigs and has not been shown to cross the placental barrier in this species (Shreeve, Patterson, Pepin et al. 1977). Similarly, Munro, Scott, Moodie & Willes (1973) found no effect in ovine foetuses even with a dose that was sufficient to cause death of the dam; the toxin was found in foetal blood only in trace amounts. Information on the distribution and excretion pattern of OCT A is limited. Studies in rats by previous workers have demonstrated accumulation of the toxin in the kidney and gastro-intestinal tissues (Chang & Chu, 1977; Lillehoj, Kwolek, Elling & Krogh, 1979; Suzuki, Satoh & Yamazaki, 1977). The autoradiographic study reported here was an attempt to gain information on the general distribution of radioactivity from ~4C-labelled OCT A in the maternal and foetal tissues of mice. A preliminary report of the results obtained was presented at the International Conference on Veterinary Pharmacology, Toxicology and Therapeutics held at Cambridge in July/August, 1980.
Abbreviations: OCT A = Ochratoxin A; OCT ct =ochratoxin c~; TLC = thin-layer chromatography.
Autoradiography. Seven C57B1 female mice (obtained from the Swedish National Defence Research Institute, Stockholm) were used for whole-body autoradiography two on day 9, four on day 10 and one on day 17 of pregnancy. The day following the night of conception, as judged from vaginal plugs, was called day 1 of gestation. The animals were killed 20 rain, 1, 3 or 4 hr after injection of labelled OCT A. Longer time intervals were not possible because of the toxicity of the dose given (see below). A further four females, one each on pregnancy days 8, 9, l0 and 17, were used for autoradiography of their dissected uteri with a survival time of 20 min after injection. [~4C]OCT A prepared by fermentation using sodium [1-~4C]acetate, was generously supplied by Dr E. B. Lillehoj, SEA, USDA, New Orleans, USA. Each of the animals was injected iv with 4.8 mg [~4C]OCT A, which corresponded to about 120~170mg/kg. The substance was dissolved in 0.2 ml 0.1 M-NaHCO3. The dose used was several times the oral LDs0, which has been found to be 29 30 mg/kg (Arora, 1982). The specific activity of the labelled OCT A was 1 x 106 dpm/mg, so that every animal received about 2#Ci (E. B. Lillehoj, personal communication). Whole-body autoradiography and autoradiography of the uteri were performed according to Ullberg (1954 & 1977) after rapid freezing of the whole animals or the dissected uteri. Fluorography. Some of the sections from the isolated uteri were taken on tape 688 (3M Co., St Paul, MN, USA) and placed under ultraviolet (UV) light. The intense light-blue fluorescence of OCT A was easily distinguishable from the natural dim-blue fluorescence of the control sections from animals free from OCT A. Liquid scintillation counting and chromatograph),. One C57B1 mouse was killed on each of days 8, 9, 10 and 17 of pregnancy, 20min after injection of
563
L.-E. APPELGRENand R. G. ARORA
564
4.8 mg [14C]OCT A. The tissue samples (liver, kidney, uterus, placenta and foetus) were weighed, homogenized and extracted with 0.01 M-H3PO 4 and with chloroform. Aliquots from the water and chloroform phases were counted in a liquid scintillation counter using Aquasol 2 (NEN, Boston, MA, USA) as scintillation medium. Thin-layer chromatography (TLC) was used to analyse the chromatographic behaviour of the radioactivity in the chloroform extracts. Benzene acetic acid (9:1, v/v) was used as the solvent system and the radioactivity was detected with the aid of a radiochromatogram scanner. The non-radioactive standard OCT A was identified by its fluorescence under UV light. For a control experiment another t4C-labelled OCT A preparation was synthesized. The side-chain of non-radioactive OCT A was split off by acid hydrolysis and ~4C-labelled phenylalanine (sp. act. 30 mCi/mmol) was introduced into the molecule. The synthesis was kindly performed by Dr Bj6rn Lindgren of the Swedish National Defence Research Institute, UmeA. Since the exchange of radioactive OCT A was very small, only one animal could be used. Thus, a 9-day pregnant CBA mouse was injected iv with all the [~4C]OCT A, corresponding to 0.05/tCi. (The specific activity is not known since it was impossible to weigh the small amount. Radiochromatography of the original compound showed only one radioactive spot with the same R~ value as the reference non-radioactive OCT A.) After 20 min, the animal was killed and tissue samples were taken. The samples were counted in a liquid scintillation counter after dissolving the samples in Soluene-350 (Packard), and addition of Insta-Fluor (Packard). Quench-correction was made using an external standard. RESULTS
A utoradiography With a 20-min delay after injection of [14C]OCT A, the highest concentration of radioactivity was found in the bile, indicating rapid biliary excretion of the mycotoxin and/or its labelled metabolites (Fig. 1). The liver showed a fairly high t4C concentration as did the pelvic part of the kidney. The salivary glands and large blood vessels showed intermediate concentrations, and the brown fat, myocardium and uterus showed slightly higher concentrations than the blood. One hour after injection, the bile still had the highest ~4C concentration, followed by the liver and kidney; a "spotty" pattern was observed in the kidney cortex (Fig. 2). The blood showed a fairly high ~4C concentration which exceeded that of the myocardium and salivary glands. In the spleen and thymus the ~4C concentration was lower than in the blood. Four hours after injection, the intestinal contents (bile) showed a very high ~4C concentration, while the liver, kidney, salivary glands and brown fat showed about the same concentration as that of the blood which was still relatively high. At this time after the injection the ~4C concentration in the thymus, the white pulp of the spleen and the corpora lutea in the ovary also reached that of the blood (Fig. 3). In the bone and central nervous system no radioactivity could be detected.
Uterus and foetus With a 20-min delay after injection of labelled OCT A to the 8- and 9-day pregnant mice, the ~4C concentration in the uterine wall was higher than that of the blood (Fig. 4a,c). A still higher concentration was seen in the ectoplacental cone and in some embryonal structures most probably corresponding to the allantois and the gut, in the 9-day pregnant mice. The corresponding 10-day pregnant animals showed less concentration in the uterine wall (Fig. 4b, d) and the placental structures, but in some embryos a high concentration of radioactivity could be detected in the allantois. In the 17-day pregnant animals, the concentration was very low in the foetuses, indicating that very small amounts of radioactivity passed the placental barrier (Fig. 5a, b). High amounts of radioactivity were found, however, in the chorio-allantoic placenta and still higher amounts in the visceral yolk sac.
Fluorograpby The blue fluorescence distribution of OCT A showed the same pattern in the pregnant uteri and embryo/foetus as was shown by autoradiography (Fig. 4). The concentration of mycotoxin in the 9-day pregnant uterus was much higher than that in the 10-day pregnant uterus; some embryonal structures showed fluorescence in the 9- but not in the 10-day pregnant mice. In the 17-day pregnant uterus the strongest fluorescence was found in the visceral yolk sac placenta.
Liquid scintillation counting and chromatography The results of the radioactivity counts in the chloroform extracts of the tissues are presented in Fig. 6, showing counts in the liver, kidney and uterus 20 min after injection of [~4C]OCT A in 8-, 9- and 10-day pregnant animals. From the 17-day pregnant animal, the placenta and foetus were analysed instead of the uterus. (The ~4C concentration in the water extracts was not above the background level in the tissue samples analysed.) There was good agreement regarding the relative concentration between these results and the autoradiographic findings. The radioactivity concentration in the uteri of the 8- and 9-day pregnant animals was higher than in the 10-day pregnant uterus. In the foetus of the 17-day pregnant animal only a small amount of ~4C was found (Fig. 6). Radiochromatography of the chloroform extracts revealed that the radioactivity in the blood as well as in the liver, kidneys, foetuses and uteri (Fig. 7) corresponded to the reference substance [~4C]OCT A. In the control experiment, using the [~4C]OCT A with theoretically much higher specific activity, the highest concentration was found in the bile, followed by the blood (Table 1). DISCUSSION
Because of the low specific activity of the compound used and the minimum dose required for autoradiographic experiments, the toxicity of OCT A did not allow the animals to survive longer than 4 hr after injection. The highest concentration of radio-
White
Liver
Kidney
I larderian
Fig. 2. Detail of whole-body [‘4C]OCT A, showing
gland
autoradiogram the “spotty”
Heart
Myocardium
Intestinal contents
(bile)
of a 17.day pregnant mouse 1hr after iv injection distribution of radioactivity in the kidney cortex.
Fig. 3. Whole-body autoradiogram of a IO-day pregnant showing a blood concentration of ‘%I comparable
Uterus
blood
pulp of spleen
mouse 4 hr after iv injection of [‘%I]OCT A, to that of liver, kidney and spleen.
Uterus
Embryo
of
Embryo
Fig. 4. OCT A fluorescence (intense light areas) in uteri taken from mice injected 20 min earlier with c. 150 mg [‘4C]OCT A/kg on (a) day 9 and (b) day IO of pregnancy. The blue fluorescence is apparent in some embryos and is much stronger in the uterus in (a) than in (b), in which it is restricted to the uteri and placentae. This distribution pattern is reflected in the autoradiograms of adjacent sections from the (c) 9-day and (d) IO-day uteri. 565
Liver : :
Fig. 5. Detail of (a) whole-body
Foetus
Yul ksac Dlacenta
: :
autoradiogram and (b) corresponding I hr after iv injection of [‘%]OCT 566
section of a 17.day pregnant A.
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Distribution of ochratoxin A
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Fig. 6. Radioactivity in chloroform extracts from the liver, kidney and uterus (placentae and foetuses) 20 min after iv Origin : ~, ! injection of ['4C]OCT A to mice at different stages of pregnancy, showing a differencein concentration in the uteri Fig. 7. Radioactive thin-layer chromatograms scanned in a between day 8 or 9 and day l0 of pregnancy (cf. Fig. 4). radiochromatogram scanner and showing one main radioactivity spot corresponding to the reference [~4C]OCTA in the chloroform extracts from livers, kidneys, uteri, placentae and tbetuses of mice injected with ['4C]OCT A. activity was found in the bile, indicating a rapid and high biliary excretion. In earlier studies, high concentrations of OCT A have been found mainly in the intestines and kidney and no biliary excretion of the toxin has been mentioned (Galtier, 1974; Lillehoj et al. 1979; Suzuki et al. 1977). Apparently, the high biliary excretion observed in the autoradiograms was not due to the very high amount of toxin administered since in our control experiment, using a tracer dose, a similar excretion pattern was observed (Table 1). Also, the concentration of ]4C in the other tissues investigated in this control experiment was of the same relative magnitude as in the autoradiograms. The blood showed a fairly high concentration 4hr after injection, a finding in agreement with the affinity of OCT A for serum albumin (Chang & Chu, 1977; Chu, 1971). The amounts of radioactivity found in the white pulp of the spleen and in the thymus 4hr after iv injection of [~4C]OCT A may be related to the lymphocidal action of OCT A in these organs (Arora, 1982; Kanisawa, Suzuki, Kozuka & Yamazaki, 1977). The "spotty" appearance of OCT A in the kidney can be related to the lesions observed in the proximal tubules (Elling et al. 1975) and to the demonstration of OCT A by immunofluorescence in the kidneys of pigs and rats (Elling, 1977). The relatively low uptake of OCT A by the uterus of the 10-day pregnant mouse compared to the 9-day animal, observed from the autoradiography as well as the fluorography studies (Fig. 4) was also confirmed by the liquid scintillation data, which showed the 10-day uterus to have considerably lower counts than either the 8- or 9-day uterus. The demonstration of a relatively high concentration of radioactivity and of a strong blue fluorescence in embryos following injection on day 9 but not in those exposed on day 10 or 17 of gestation is in agreement with the observed high frequency of malformations in foetuses of dams given OCT A on day 9 of gestation (Arora & Fr616n, 1981). The low concentration of radioactivity registered in the embryo/foetus after injection
on day 10 or 17 supports the theory that the amount crossing the placental barrier on or after day 10 of development is perhaps insufficient to induce morphological abnormalities (Arora & Fr616n, 1981). Earlier studies showed that the toxin did not pass the placental barrier in swine (Shreeve et al. 1977) and it was found only in trace amounts in the foetal blood after iv injection into pregnant sheep (Munro et al. 1973). In the present experiment the slight radioactivity found in the corpora lutea 4hr after injection can be correlated with interference of OCT A with steroidogenesis, as reported from histochemical studies in rats (Gupta, Bandyopadhyay, Mazumdar & Paul, 1980). Even though the radioactivity demonstrated in the autoradiograms could be due to metabolites of the injected compound, the chromatographic studies have shown that the radioactivity in the investigated organs corresponded to the reference substance [~4C]OCT A. Similar results were obtained in rats dosed ip, since OCT A but not its metabolite was found in the blood, liver and kidney (Nel & Purchase, 1968). However, studies using [14C]OCT A have shown that urine and faeces from orally dosed rats contained OCT A and ochratoxin ~ (OCT ~) (Suzuki et al. 1977). Homogenates of pancreas, duodenum and ileum, but not liver and kidney, have been shown Table 1. t¢C concentration in various organs and body fluids from a 9-day pregnant mouse Tissue/fluid Bile Blood Uterus Kidney Liver Muscle (skeletal)
14C concentration (dpm/g tissue × 10 3) 34.0 5.5 1.7 2.5 2.6 0.3
The animal was killed 20 min after iv injection of 0.05 #Ci [14C]OCT A (theoretical sp. act. 30 mCi/mmol).
L.-E. APPELGRENand R. G. ARORA
568
by the same authors to hydrolyse OCT A to OCT ~, indicating that this t r a n s f o r m a t i o n is probably greater in orally dosed animals than in parenterally injected animals. Acknowledgements--This work was supported by the Swedish Council for Forestry and Agricultural Research (SJFR) and the Swedish Council for Planning and Coordination of Research (heavy equipment). REFERENCES
Arora R. G. (1982). Some observations on the pathological effects of ochratoxin A in adult and foetal mice (Abstract). Toxicon 20, Suppl. 1, p. 3. Arora R. G. & Fr61~n H. (1981). Interference of mycotoxins with prenatal development of the mouse. II. Ochratoxin A induced teratogenic effects in relation to the dose and stage of gestation. Acta vet. scand. 22, 535. Arora R. G., Fr616n H. & Nilsson A. (1981). Interference of mycotoxins with prenatal development of the mouse. I. Influence of aflatoxin B~, ochratoxin A and zearalenone. Acta vet. stand. 22, 524. Chang F. C. & Chu F. S. (1977). The fate of ochratoxin A in rats. Fd Cosmet. Toxieol. 15, 199. Chu F. S. (1971). Interaction of ochratoxin A with bovine serum albumin. Archs Biochem. Biophys. 147, 359. Chu F. S. (1974). Studies on ochratoxins. C R C Crit. Rev. Toxicol. 2, 499. Elling F. (1977). Demonstration of ochratoxin A in kidneys of pigs and rats by immunofluorescence microscopy. Acta path. microbiol, scand. Sect A 85, 151. Elling F., Hald B., Jacobsen Chr. & Krogh P. (1975). Spontaneous toxic nephropathy in poultry associated with ochratoxin A. Acta path. microbiol, scand. Sect. A 83, 739. Galtier P. (1974). Devenir de l'ochratoxine A dans
l'organisme animal. II. Distribution tissulaire et 61imination chez le rat. Annls Rech V~t. 5, 319. Gupta M., Bandyopadhyay S., Mazumdar S. K. & Paul B. (1980). Ovarian steroidogenesis in rats following ochratoxin A treatment. Toxic. appl. Pharmac. 53, 515. Hood R. D. (1979). Effects of mycotoxins on development. In Advances in the Study o f Birth Defects. Vol. II. Edited by T. V. N. Persaud. p. 191. MTP Press, Lancaster. Kanisawa M., Suzuki S., Kozuka Y. & Yamazaki M. (1977). Histopathological studies on the toxicity of ochratoxin A in rats. I. Acute oral toxicity. Toxic. appl. Pharmac. 42, 55. Krogh P., Hald B. & Pedersen E. J. (1973). Occurrence of ochratoxin A and citrinin in cereals associated with mycotoxic porcine nephropathy. Aeta path. microbiol. scand. Sect. B 81, 689. Lillehoj E. B., Kwolek W. F., Elling F. & Krogh P. (1979). Tissue distribution of radioactivity from ochratoxin A~4C in rats. Mycopathologia 68, 175. Munro I. C., Scott P. M., Moodie C. A. & Willes R. F. (1973). Ochratoxin A--Occurrence and toxicity. J. Am. vet. Med. Ass. 163, 1269. Nel W. & Purchase I. F. H. (1968). The fate of ochratoxin A in rats. J. S. Afr. Chem. Inst. 21, 87. Shreeve B. J., Patterson D. S. P., Pepin G. A., Roberts B. A. & Wrathall A. E. (1977). Effects of feeding ochratoxin to pigs during early pregnancy. Br. vet. J. 133, 412. Suzuki S., Satch T. & Yamazaki M. (1977). The pharmacokinetics of ochratoxin A in rats. Jap. J. Pharmac. 27, 735. Ullberg S. (1954). Studies on the distribution and fate of 35S-labelled benzyl-penicillin in the body. Acta radioL Suppl. 118, p. 1. Ullberg S. (1977). The technique of whole body autoradiography. Cryosectioning of large specimens. The L K B Instrument Journal. Special issue on whole body autoradiography, Sweden. p. 1.